Project Summary Macrophage-mediated programmed cell removal (PrCR) allows clearance of living cells. We have shown that this phagocytic process can eliminate cancer cells that present an ?eat me? signal and have their dominant 'don't eat me' molecules blocked. We then further extended this to the clearance of other pathogenic or ?expired? cells. The key novel findings of our recent studies are: 1. Activated macrophages produce and secrete calreticulin (CRT); 2. Secreted CRT binds to surface asialoglycans on target cells to create ?eat me signals' for macrophages. 3. Cancer cells and neutrophiles modulate their surface to expose asialoglycan binding sites for CRT that acts as an ?eat me? signal for macrophages. We propose that by binding both to asialoglycans and to pro-phagocytic receptors on macrophages (CD91/LPR1), CRT can bridge target cells to macrophages for clearance via PrCR. CRT is normally a resident ER protein containing a C-terminal KDEL retention signal, but it?s been shown that in dying cells CRT can be translocated to the cell surface. We found that upon macrophage activation via toll-like receptors (TLR), CRT can both translocate to the cell surface and be secreted, leading to increased PrCR of either WT (dying) or Bcl-2+ (viable) peritoneal neutrophils and cancer cells on which the ?don?t eat me? signal CD47 is either absent or blocked. Based on these findings we proporse: (1) to elucidate the signals affecting the macrophage that result in CRT translocation to the cell surface and secretion of soluble forms of CRT; (2) to elucidate the mechanisms regulating the availability of asialoglycan-containing binding sites for CRT on target cells. Elucidating the individual mechanisms in macrophages and target cells required for PrCR and understanding the cross-talk within macrophage:target-cell interaction can have broad therapeutic implications. In Aim 1 we will investigate which signals stimulate macrophages to increase cell surface expression and secretion of CRT for PrCR and the heterogeneity of macrophages that can carry out PrCR in vitro and sterile inflammation in vivo. In Aim 2 we will employ proteomic analysis to define and characterize the different proteoforms of CRT originating in macrophages before or after stimulation: the ER form vs. cell-surface- bound, vs. soluble secreted CRT, vs. CRT that is bound to asialoglycans on target cells. We have preliminary evidence that the secreted form of CRT does not contain the KDEL motif and that potential proteolytic processing leads to the formation of the different CRT forms. Lastly in Aim 3 we will study the initiating signaling events and enzymes that affect the addition or removal of sialic acid, the activity of which determines the level of exposed asialoglycans and thus the binding of CRT to the surface of cells destined for elimination. These studies will shed light on a novel mechanism by which macrophages detect cells that are to be removed from the body. Our findings will have a broad relevance to cancer, degenerative disease, and inflammatory lesions and will likely reveal new therapeutic targets for life altering disease conditions.